Mutation breeding techniques can be applied to develop new germplasm having desirable agronomic characteristics. Mutation breeding is initiated by generating a Population of plants with increased genetic variability through mutagenesis. The resulting population is then subjected to selective conditions which identify individuals expressing a desired trait or characteristic. Gottshalk et al., Induced Mutations in Plant Breeding, (Springer-Verlag, New York, 1983, PP. 16-17), and Simmonds, Principles of Crop Improvement, (Longman, N.Y., 1979, pp 297-303) are general references covering various mutation breeding techniques.
Mutations created using such procedures can occur at widely different frequencies, with highly desired traits often induced at extremely low rates. Where mutation frequencies are reasonably high, screening of the population to identify new traits has been accomplished by (1) exposure of progeny of the mutagenized population to a chemical treatment which kills unmodified specimens, or (2) inspection of plants germinated from the mutagenized seed for evidence of the new property. For example, Pinthus et al., Science. 177:715 (1972) described mutation breeding experiments involving herbicides. In these experiments, M.sub.2 seed Populations of tomato and wheat were generated by soaking wheat and tomato seeds in 8 mM ethyl methanesulfonate (EMS). The treated seeds were then screened by sowing in soil containing the herbicides diphenamid or terbutryn at concentrations inhibitory to growth of the normal parental variety. The tolerant tomato lines obtained showed a 25% reduction in seedling weight in response to treatment with diphenamid, while the original cultivar exhibited a 40% reduction in seedling weight. Increased tolerance to herbicide was also reported for certain mutant wheat lines, although quantitative results were not provided.
Raut et al., Indian J. Genet. Plant Breed, 42:265-270 (1982), utilized chemical mutagenesis to induce and select mutations for a different seed coat color. Chamberlain and Bernard, Crop Science, 8:728-729 (1968) report the failure to obtain resistance to brown stem rot through mutagenesis despite the fact that such resistance can be found in nature.
Agrichemical Age, June 1984, p. 20, reports that USDA scientists W. L. Barrentine and E. E. Hartwig discovered variation with respect to tolerance for the herbicide metribuzin within the soybean cultivar Glycine max vc. "Tracy". Tolerant individuals were present at approximately a 5% frequency in the "Tracy" population, and were not mutants resulting from mutation breeding techniques.
There have been no reports of mutant soybean plants with altered acetolactate synthase (ALS) function associated with herbicide resistance.
Where mutations are extremely rare, many more seeds must be mutagenized and screened to obtain the new trait. It is well known that dominant mutations occur much less frequently than recessive mutations. In fact, to date, no one has ever selected a dominant mutation in soybean. Gottschalk et al., in their extensive review of mutation breeding, state that "about 1% of all induced mutations are dominant ones". This means that approximately 100 times as many individuals must be screened to find a dominant mutation as to find a recessive mutation. Using Arabidopsis thaliana as a model system, (Somerville, Trends in Genetics. 2: 89-93 (1986)) states that "for many loci, a mutation resulting in loss of function" (which is generally synonymous with a recessive mutation) "can be recovered by screening approximately 2000 M2 plants". Using these sources as guidelines, isolation of a dominant mutation in Arabidopsis could conceivably require screening of at least 200,000 M2 individuals depending on the locus in question and the vagaries of sampling the M2 population. The search for a dominant mutation, therefore, requires 100 times the work of a search for a recessive mutation. Such experiment size or labor is totally impractical. Due to the minute seed size of Arabidopsis thaliana, Somerville (Mol. Gen. Genet. (1986) 204: 430-434) was, however, able to isolate dominant chlorsulfuron-resistant mutants by screening up to 10,000 M2 seeds on a single 90 mm petri plate. Hardcastle [Hardcastle W. S. 1979. Soybean (Glycine max) cultivar response to metribuzin in solution culture. Weed Science (27):278-279] discloses the use of hydroponics to study the response of small numbers of soybean cultivars to a single concentration of metribuzin. Feenstra and Jacobsen, (TAG 58:39-42) disclose the use of hydroponics to select a recessive pea mutant lacking nitrate reductase (NR) activity. Their selection system consisted of growing seedlings for 7 days in moist vermiculite, removing the cotyledons, and trans- planting the seedlings to a small pan (22.5.times.22.5.times.5.5 cm) containing vermiculite and a nutrient solution. Five days later (when the plants were 12 days old), the nutrient solution was eventually supplemented with a chlorate solution. Since NR reduces chlorate to chlorite, which is toxic to plants, plants lacking NR could be selected based on their lack of chlorate damage. Since the plants were not treated with chlorate until they were seedlings of considerable age (12 days) and size, plant spacing to permit normal growth and visual observation must have limited the density (Plants Per unit area) at which seedlings were screened. Feenstra and Jacobsen screened 12 M2 seedlings from each of 1090 fertile plants. Although this procedure enabled them to screen enough M2 plants (roughly 13,080) to find one recessive NR mutant, transplanting and plant spacing would make it extremely laborious and space consuming to screen the population size required to find a dominant mutation.
One highly desirable agronomic characteristic currently sought in elite, commercial germplasm of a number of crop plants is true herbicide resistance. If selected within an elite soybean cultivar, a dominant soybean mutation conferring herbicide resistance could be immediately incorporated into an agronomic breeding program. Such mutations would be expected to be extremely rare, however, and no dominant mutation of any type has been discovered through seed mutagenesis and reported for soybean. Specifically, a dominant mutation that decreased the sensitivity of the target enzyme acetolactate synthase (ALS) to compounds that are herbicidal due to the inhibition of ALS was desired. Such resistance would require a specific change that renders the ALS enzyme resistant to the herbicide to insure that a cultivar would sustain little or no injury when exposed to the herbicidal compound, either in screening operations or in the field for weed control. It has been found that seed mutagenesis and mutant selection from a population of up to one million seeds is required to isolate a dominant, herbicide resistant plant mutation. A clear need exists for such dominant mutant plants as a basis to develop commercial soybean cultivars efficiently without sacrificing existing agronomic traits.